蜡样芽孢杆菌Bacillus cereus LJ01中亚硝酸盐还原酶的基因克隆、表达和纯化

Cloning, Expression and Purification of the Nitrite Reductase Gene from Bacillus cereus LJ01

利用克隆表达技术,对一种蜡样芽孢杆菌Bacillus cereus LJ01的亚硝酸盐还原酶(nitrite reductase,Ni R)基因进行克隆、原核表达,利用Ni柱亲和层析和DEAE Sepharose Fast Flow交换层析对表达的重组NiR进行纯化,分析重组酶的性质。研究结果显示,该NiR含有一个1 623 bp的开放阅读框,编码540 个氨基酸序列,重组NiR的分子质量约为67 ku;该酶中同时存在铁离子和铜离子,且含量分别为51.0 mg/kg和184.5 mg/kg;圆二色谱结果显示α-螺旋结构在重组NiR中所占比例最大。

A nitrite reductase(NiR)gene was cloned from Bacillus cereus LJ01and expressed in Escherichia coli.The recombinant enzyme was purified by His-Tag nickel affinity chromatography(Ni Sepharose6Fast Flow)and DEAE Sepharose Fast Flow ion exchange chromatography for characterization.The results showed that the recombinant NiR gene contained a1623bp-length ORF encoding540amino acids,and the molecular weight was about67ku.Iron and copper ions were simultaneously present in the enzyme and their contents were51.0and184.5mg/kg,respectively.The results of circular dichroism showed that the helix structure accounted for the largest proportion of the recombinant NiR.